A new species and new records of Hymenopellis and Xerula (Agaricales, Physalacriaceae) from China

Hymenopellis is the genus that exhibits the highest number of species within the Xerula/Oudemansiella complex. Numerous species of Hymenopellis demonstrate edibility, and some of these species have been domesticated and cultivated. During an extensive survey carried out in Henan and Jilin Provinces, China, a substantial quantity of Hymenopellis specimens was gathered as a component of the macrofungal resource inventory. Based on the findings of morphological and molecular phylogenetic studies, a new species, Hymenopellis biyangensis, has been identified. A new record species, Hymenopellis altissima, has been discovered in China. Additionally, two new record species, Hymenopellis raphanipes and Xerula strigosa, have been found in Henan Province. Internal transcribed spacer (ITS) and large subunit ribosomal (nrLSU) were used to establish a phylogeny for species identification. Detailed descriptions, field habitat maps and line drawings of these species are presented. The discussion focuses on the relationships between newly discovered species and other related taxa. Additionally, this study provides and a key to the documented species of Hymenopellis and Xerula found in China.

In the 21st century, researchers have demonstrated, based on the finding of morphological and molecular systematics, that Oudemansiella and Xerula are two independent genera.Furthermore, the numerous species that were previously classified under Xerula should now be reclassified under Oudemansiella (Redhead, 1987;Wang et al., 2008).Zhang (2006) showed that the Oudemansiella may be divided into three groups: sect.Oudemansiella, sect.Dactylosporina and sect.Radicatae based on molecular phylogenetic studies constructed from (ITS) and (nrLSU).Wang et al. (2008) redefined the genus Xerula s. str.and providing characterizations of the genus.Yang et al. (2009) excluded the narrowly defined genus Xerula from the genus Oudemansiella and categorized the genus Oudemansiella into four groups: sect.Oudemansiella, sect.Mucidula, sect.Dactylosporina and sect.Radicatae.Petersen & Hughes (2010) revised the taxonomic relationships of the Xerula/Oudemansiella complex based on morphological evidence and molecular phylogenetic results from ITS and nrLSU.They proposed 68 new taxa, including four new genera (Hymenopellis, Paraxerula, Pointiculomyces, Protoxerula), accepting the definition of the genus Xerula from Yang et al. (2009), these results have been widely accepted and cited in subsequent studied (He et al., 2019;Niego et al., 2021;Park et al., 2017).
During a survey of macrofungal resources in the Henan and Jilin Provinces of China, we collected some specimens of Hymenopellis.After conducting a comprehensive study that combins morphological and phylogenetic analysis, we the identification of a new species, a new record from China and two new records from Henan.These findings contribute to the overall species diversity within the taxon and provide valuable molecular data for further research in this field.

Sampling and morphological analysis
The specimens were gathered from Biyang County, Henan Province and Shulan City, Jilin Province, China.Voucher specimens were deposited in the Herbarium of Mycology of Jilin Agricultural University (HMJAU).Macroscopic morphological characteristics are derived from field observations of fresh specimens, while microscopic morphology is examined using a light microscope.The primary reagents utilized in the analysis included a 5% solution of potassium hydroxide (KOH), a 1% solution of Congo red solution, and Meler's reagent solution.The colors of basidiomata were described using the color coding system developed by Kornerup and Wanscher's (Kornerup & Wanscher, 1978).The size of basidiospores is expressed as (a) b-c (d), where ''a'' is the minimum and ''d'' is the maximum, and 95% of the observed range falls between b-c.''Q'' is the ratio of the length to width of the basidiospores, and ''Q ± av'' is the average Q ± standard deviation of all basidiospores (Dong & Bau, 2022;Wang et al., 2022).

DNA extraction, PCR amplification, and sequencing
The total DNA was extracted from dried specimens using the NuClean Plant Genomic DNA kit (Kangwei Century Biotechnology Co., Ltd., Beijing, China).Primers for amplification of ITS were ITS 4 and ITS 5 (He et al., 2023;Ward & Akrofi, 1994), nrLSU were LROR and LR5 (Kauserud & Schumacher, 2001;Wang & Yang, 2023).The PCR program was as follows: pre-denaturation at 94 • C for 5 min; then followed by 35 cycles of denaturation at 94 • C for 50 s, annealing at 50 • C (ITS and nrLSU) for 50 s, and elongation at 72 • C for 70 s.Finally, a final elongation at 72 • C for 8 min was included.Then, the PCR products were sent to Biotechnology Co., Ltd. in Shanghai, China for sequencing.

Phylogenetic analysis
The news sequences were uploaded on NCBI (http://www.ncbi.nlm.nih.gov).ITS and nrLSU sequences of related taxa were retrieved from GenBank and related articles (Hao et al., 2016;Lin et al., 2021;Matheny et al., 2006;Niego et al., 2021;Petersen & Hughes, 2010;Qin et al., 2014).The sequences obtained in this study, along with those of related taxa, are listed in Table 1.The ITS and nrLSU datasets include 109 sequences from 33 species of 7 genera within the Physalacriaceae family.Flammulina yunnanensis ZW Ge & Zhu L. Yang and Flammulina velutipes (Curtis) Singer were selected as outgroups for the phylogenetic analysis of the Hymenopellis, Paraxerula americana (Dörfelt) RH Petersen and Paraxerula hongoi (Dörfelt) RH Petersen were selected as outgroups for the phylogenetic analysis of Xerula.
The sequences from the two datasets were compared and manually modified separately using BIOEDIT (Hall, Biosciences & Carlsbad, 2011;Ye & Bau, 2022).PartitionFinder 2 was used to determine the optimal model scheme for two multi-locus datasets (Lanfear et al., 2017).Maximum parsimony analysis was applied to a combined dataset of two genera, following the approaches described by Li, Zhao & Liu (2022).Phylogenetic analyses of the two genera were performed using the maximum likelihood (ML) and Bayesian inference (BI) methods, respectively.The maximum likelihood method (ML) was performed using IQTree 1.6.8(Nguyen et al., 2015), with ultrafast bootstrapping and 5,000 repetitions.The Bayesian inference (BI) was performed using MrBayes 3.2.6 (Deng et al., 2022;Ronquist et al., 2012)

Molecular phylogeny
The two-locus gene dataset (ITS + LSU) of Hymeopellis contains 51 sequences and 1,770 aligned characters in length.Out of these, 1,242 characters are constant, 439 characters are parsimony-informative, and 89 characters are variable and parsimony-uninformative.For the multi-locus data set of Hymenopellis, the optimal model for ITS was HKY + F + I + G4 (Liao et al., 2022), and the optimal model for nrLSU was K2P + I (Ripplinger & Sullivan, 2008).Because the phylogenetic trees generated from the combined dataset using ML and BI analyses exhibited nearly identical topology, we have chosen to display only the ML tree (Fig. 1).In the phylogenetic tree, H. biyangensis and H. altissima are belonged to the genus Hymenopellis, and each forms a well-supported branch (BP = 99, PP = 1 and BP = 99, PP = 1).
Hymenopellis raphanipes collected from Biyang County, China, is found on the same branch as Hymenopellis raphanipes distributed in two other locations (Fig. 1), and it received strong support (BP = 100, PP = 1).
Habitat.Solitary or scattered on the soil of mixed forests of Quercus robur and Torrey pine.
Habitat.Growing on the ground in mixed broad-leaved forests dominated by Quercus robur.

DISCUSSION
In this study, a new species, Hymenopellis biyangensis, was discovered, along with a new record species of H. altissima for China.Additionally, two new record species, H. raphanipes and X. strigos a were found in Henan province through the use of morphology and molecular phylogeny.
In the phylogenetic analysis, the new species H. biyangensis formed an independent clade and was found to be a sister group with H. altissima, which received high support (BP = 99, PP = 1) (Fig. 1).However, the pileipellis of H. altissima has an ixohymeniderma while H. biyangensis does not.In addition, H. biyangensis and H. altissima can be distinguished by their pleurocystidia and basidiospores.H. biyangensis have paddle-shaped, obovoid pleurocystidia and ellipsoid to broadly ellipsoid basidiospores, while H. altissima has fusiform, obviously capitulates pleurocystidia and globose to subglobose basidiospores.
Pleurocystidia of Hymenopellis species are mostly described as ''utriform'', ''jar-shaped'' and ''ten pin-shaped'' (Petersen & Hughes, 2010), while H. biyangensis has broadly clavate, paddle-shaped to obovoid pleurocystidia.In addition, both H. biyangensis and H. altissima are relatively rare 2-spore species in the genus Hymenopellis.Among the 2-spored species, the basidiospores of H. altissima are subglobose to globose different from the other species.Hymenopellis biyangensis, H. altissima and H. raphanipes are similar in habitat, growing on the forest dominated by Fagaceae.In the description by Hao (2016), Petersen & Hughes (2010), thirteen species of Hymenopellis that were found to grow in Fagaceae forests, particularly in Fagus and Quercus.This suggests that there may be a relationship between the growth of species in this genus and Fagaceae, which should be further studied.
Hymenopellis is a paraphyletic taxon (Vellinga, 2010) and represents a group of morphologically similar taxa that were previously distributed in several sections of Oudemansiella (Petersen & Hughes, 2010).Therefore, some researchers have also questioned the establishment of Hymenopellis.Hao (2016) proposed a new taxonomic system in which Oudemansiella is recognized as a highly supported monophyletic lineage, and should be treated as a single genus.A new systematic arrangement with three sections, namely, sect.Oudemansiella, sect.Dactylosporina and sect.Radicatae has been proposed; Hymenopellis should be subsumed into the genus Oudemansiella (Hao, 2016).Therefore, more researches on this taxon is still needed in the future.
• Xin-yue Gui performed the experiments, analyzed the data, prepared figures and/or tables, and approved the final draft.
• Peng Dong performed the experiments, prepared figures and/or tables, and approved the final draft.
• Yang Wang analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.
• Bo Zhang conceived and designed the experiments, authored or reviewed drafts of the article, and approved the final draft.
• Xiao Li conceived and designed the experiments, authored or reviewed drafts of the article, and approved the final draft.

Table 1 (
continued) *Sequence retrieved from GenBank.The newly generated sequences in this study are indicated in bold.
The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants, and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone.In addition, new names contained in this work have been submitted to MycoBank from where they will be made available to the Global Names Index.The unique MycoBank number can be resolved and the associated information viewed through any standard web browser by in PhyloSuite1.2.2 (Zhang et al., 2020), running for 2,000,000 generations.The initial 25% of the sampled data was discarded as burn-ins.Nomenclature''appending the MycoBank number contained in this publication to the prefix ''http: //www.mycobank.org/MycoTaxo.aspx?Link=T&Rec=''.The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central SCIE, and CLOCKSS.''(https://peerj.com/about/policies-and-procedures/#new-species).